CN107843736A - A kind of collaurum early pregnancy detection reagent and preparation method thereof - Google Patents
A kind of collaurum early pregnancy detection reagent and preparation method thereof Download PDFInfo
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- CN107843736A CN107843736A CN201710833041.2A CN201710833041A CN107843736A CN 107843736 A CN107843736 A CN 107843736A CN 201710833041 A CN201710833041 A CN 201710833041A CN 107843736 A CN107843736 A CN 107843736A
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- collaurum
- antibody
- nano gold
- detection reagent
- early pregnancy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
Abstract
The invention discloses a kind of collaurum early pregnancy detection reagent, the collaurum early pregnancy detection reagent is mainly made up of two parts, is the golden standard liquid for linking primary antibody and secondary antibody respectively；The invention also discloses a kind of preparation method of collaurum early pregnancy detection reagent, the BSPP modifications of preparation, nano gold spherical mainly including following nano gold spherical and three steps of antibody modification of nano gold spherical；The present invention is tentatively modified collaurum using the molecule with sulfydryl, the superpower golden standard liquid of stability is made, collaurum links primary antibody and secondary antibody respectively simultaneously, respectively obtain golden standard liquid one and golden standard liquid two, using golden standard liquid stability made from the method it is high, be swift in response, high sensitivity, not consume a large amount of test paper consumptive materials, cost low, can Cord blood, freeze thawing effect is good.
Description
Technical field
The present invention relates to early pregnancy technical field of measurement and test, more particularly to a kind of collaurum early pregnancy detection reagent and its preparation side
Method.
Background technology
Gold is extracted first as heavy metal material in B.C. 5th century, when gold is divided into less than 100 nm sizes
Golden nanometer particle gold when become more precious.Golden nanometer particle (gold nanoparticle) refers to the molecule of gold, its
Diameter has higher cloud density, dielectric property and catalytic action in 1~100 nm, golden nanometer particle, has good
It biocompatibility, can be combined with a variety of large biological molecules, and not influence its bioactivity.The Jenner grain of rice of the diameter in Nano grade
Son, elementary cell are all the particles of microsize, have the unexistent physical characteristic of many macroscopic view gold, such as optical effect, table
Face effect, small-size effect, Dielectric confinement effect, macro quanta tunnel effect and some other special effectses.Gold nano
The special effects of particle causes golden nanometer particle to be widely used in the research fields such as material, medical test, disease treatment.
Collaurum is a kind of conventional labelling technique, is to be applied to the one of antigen-antibody using collaurum as tracer label thing
The new immunolabelling technique of kind, there is the advantages of its is unique.In recent years widely used in various biological studies.Make in clinic
Immunoblot assay is nearly all using its mark；Simultaneously in streaming, Electronic Speculum, immune, molecular biology so that biochip
In all may example use.1971 are that collaurum is introduced immunochemistry by Faulk and Taytor, hereafter immune colloidal gold technique
As a kind of new immunological method, increasingly extensive application has been obtained in biomedical each field.Collaurum except with albumen
Beyond matter combines, it can also be combined with many other large biological molecules, such as SPA, PHA, ConA.According to some things of collaurum
Rationality shape, such as high electron density, granular size, shape and color reaction, plus the immune and biological characteristics of conjugate, thus
Collaurum is set to be widely used in the fields such as immunology, histology, pathology and cell biology.
Human chorionic gonadotrophin（HCG）It is a kind of glycoprotein hormone as caused by placenta in human female of becoming pregnant, it is early
Pregnant Test paper uses double-antibody sandwich one step technique, using collaurum as cue mark, detects the HCG concentration in urine, comes
Make a definite diagnosis whether women becomes pregnant.Early pregnancy test paper in use, generally drops in urine in the detection hole on test paper, such as in test paper
Check plot occur a color band（The aobvious red of some test paper, the aobvious blueness of some test paper are common for red）, represent negative,
Illustrate not to be pregnant；Conversely, such as there is obvious colour band in detection zone, then it represents that it is positive, illustrate pregnant.It is most common at present
Early pregnancy test paper is the method using double antibody, is formed by combining using nitrocellulose filter, gold standard pad and sample pad, realizes people's suede
The detection of Chorionic Gonadotropin.
Conventional early pregnancy detection product is all existing in the form of test strips, and the preservation of test strips needs certain bar
Part, test strips occur damaging and are denatured the result that can all influence detection, while test strips its production needs a variety of consumptive materials and high
Expensive production equipment.
The content of the invention
The present invention is namely based on above-mentioned deficiency, proposes a kind of collaurum early pregnancy detection reagent and preparation method thereof, the present invention
Collaurum is modified using the molecule with sulfydryl, the superpower golden standard liquid of stability is made, while collaurum links one respectively
Anti- and secondary antibody, respectively obtains golden standard liquid one and golden standard liquid two, using golden standard liquid stability is high made from the method, be swift in response,
High sensitivity, not consume a large amount of test paper consumptive materials, cost low, can Cord blood, freeze thawing effect is good.
To reach above-mentioned technical purpose, present invention employs a kind of collaurum early pregnancy detection reagent, the collaurum early pregnancy
Detection reagent is mainly made up of two parts, is the golden standard liquid for linking primary antibody and secondary antibody respectively.
Preferably as the present invention, the golden standard liquid of the link primary antibody and secondary antibody is respectively golden standard liquid one and golden standard liquid two.
On the basis of the above, the invention also discloses a kind of preparation method of collaurum early pregnancy detection reagent, mainly include
Following steps：
（1）The preparation of nano gold spherical：The used glass apparatus of experiment uses chloroazotic acid（Hydrochloric acid：Nitric acid=3:1）Immersion 4 hours,
It is stand-by after repeatedly rinsing well, by 0.8-1.0m l%（Mass fraction）Gold chloride is added in 100ml ultra-pure waters, is heated to boiling
State is risen, 2.0-2.4ml 1% is then rapidly added under quick stirring（Mass fraction）Three citric acid monohydrate sodium, hold
Continuous stirring 15-20mins, obtains claret nano gold spherical solution；
（2）The BSPP modifications of nano gold spherical：100ml round-bottomed flask is taken, the nano gold spherical solution that will be cooled to room temperature adds it
In, then add 500-1000 times of BSPP, under room temperature condition, stand overnight, that is, obtain the stable nano gold sphericals modified of BSPP
Solution；
（3）The antibody modification of nano gold spherical：The BSPP nano gold sphericals modified are utilized into supercentrifuge, 10-20mins is centrifuged, removes
Fall liquid, be melted into the SDS solution of mass fraction 0.5%, be divided into two parts again, with antibody and nano gold spherical according to 100:1-1000:
1 ratio is added primary antibody and secondary antibody respectively, is then gradually added into sodium chloride solution to 0.1M is finally reached, then places 3-
5 hours, centrifugation are removed antibody unnecessary in solution, are redissolved in 0.2X TBE solution, that is, obtain the anti-of nano gold spherical
Body modifies solution.
Preferably, the step（1）Middle gold chloride mass fraction is 1%, and the mass fraction of the three citric acid monohydrates sodium is
1%。
Preferably, the step（3）In, the centrifugal rotational speed of centrifuge is 5000-8000rcf.
After above-mentioned preparation method, the invention has the advantages that：
（1）A kind of overstable collaurum early pregnancy detection reagent of the present invention, has extraordinary stability, can bear tens of
Property of the secondary Frozen-thawed cycled without destroying antibody and nano gold spherical composite modified BSPP；
（2）After the nano gold spherical of two parts of antibody modifications is mixed, it is solution occur to gather to add pregnant woman's urina sanguinis, 3-6mins or so
Collection, you can display early pregnancy tests positive；
（3）The nano gold spherical of two kinds of antibody modifications of the present invention, is separately stored in two small-sized water white transparency containers, low temperature cold
Freeze and preserve, melted using preceding room temperature, 1-2 drops are then added dropwise respectively in mixing in pan, mixes, adds testing sample, you can inspection
The HCG of test sample product is positive or negative；
（4）The present invention antibody modification nano gold spherical, stability is good, is quick on the draw, and is easy to preserve, at the same save the manufacturing into
This, energy-saving and emission-reduction.
Brief description of the drawings
Shown in Fig. 1 is the use process steps figure of product of the present invention.
Embodiment
Using embodiment, the present invention is further illustrated below.
A kind of collaurum early pregnancy detection reagent, the collaurum early pregnancy detection reagent are mainly made up of two parts, are respectively
The golden standard liquid of primary antibody and secondary antibody is linked, the golden standard liquid for preferably linking primary antibody and secondary antibody is respectively to drip golden standard liquid one and golden standard liquid two,
It developed below and specifically tells about.
The collaurum early pregnancy detection reagent of the present invention is mainly made up of two parts, is the gold mark for linking primary antibody and secondary antibody respectively
Liquid, golden standard liquid stability is high, can bear thawing without destroying golden target performance.When in use, a drop gold is added dropwise respectively
Standard liquid one and golden standard liquid two are in specifying in small-sized transparent groove, then drop pregnant woman urine to be measured is added dropwise, it is observed that probably about 3-5
After mins, you can the aggregation it was observed that red, transparent liquid reacts, become colourless transparent liquid.The present invention is double using two hydrations
(to sulfonyl-phenyl) Phenylphosphine di-potassium（BSPP）Freshly prepd nano gold spherical is modified, the collaurum being stabilized
Liquid, secondary modification then is being carried out to collaurum respectively using primary antibody and secondary antibody, is obtaining golden standard liquid one and golden standard liquid two, Jin Biao
Liquid one and golden standard liquid two are dissolved using 1%SDS buffer solutions, strengthen stability.The product stability of the present invention is extremely strong, can
The higher external interference of resistance, while this product can resist freeze-thaw damage for several times, larger change does not occur for the performance of itself, no
Influence testing result.The present invention can be used directly, easy to operate, be swift in response sensitive, and cost is low, be led in collaurum quick detection
Domain has great application value.
On the basis of the above, the invention also discloses the preparation method of collaurum early pregnancy detection reagent, below by several
Embodiment is illustrated（Incorporated by reference to Fig. 1）.
Embodiment 1
A kind of preparation method of collaurum early pregnancy detection reagent, mainly comprises the following steps：
（1）The preparation of nano gold spherical：The used glass apparatus of experiment uses chloroazotic acid（Hydrochloric acid：Nitric acid=3:1）Immersion 4 hours, it is more
It is secondary rinse well after, it is stand-by, 0.8-1.0ml gold chlorides are added in 100ml ultra-pure waters, are heated to fluidized state, Ran Hou
2.0-2.4ml three citric acid monohydrate sodium are rapidly added under quick stirring, 15-20mins is persistently stirred, obtains claret
Nano gold spherical solution；
（2）The BSPP modifications of nano gold spherical：100ml round-bottomed flask is taken, the nano gold spherical solution that will be cooled to room temperature adds it
In, then add 500-1000 times of BSPP, under room temperature condition, stand overnight, that is, obtain the stable nano gold sphericals modified of BSPP
Solution；
（3）The antibody modification of nano gold spherical：The BSPP nano gold sphericals modified are utilized into supercentrifuge, 10-20mins is centrifuged, removes
Fall liquid, be melted into the SDS solution of mass fraction 0.5%, be divided into two parts again, with antibody and nano gold spherical according to 100:1-1000:
1 ratio is added primary antibody and secondary antibody respectively, is then gradually added into sodium chloride solution to 0.1M is finally reached, then places 3-
5 hours, centrifugation are removed antibody unnecessary in solution, are redissolved in 0.2X TBE solution, that is, obtain the anti-of nano gold spherical
Body modifies solution.
In the present invention, it is preferred to step（1）Middle gold chloride mass fraction is 1%, preferably the quality of three citric acid monohydrate sodium point
Number is 1%.
In the present invention, it is preferred to step（3）In, the centrifugal rotational speed of centrifuge is 5000-8000rcf.
Embodiment 2
A kind of preparation method of collaurum early pregnancy detection reagent, mainly comprises the following steps：
（1）The preparation of nano gold spherical：The used glass apparatus of experiment uses chloroazotic acid（Hydrochloric acid：Nitric acid=3:1）Immersion 6 hours, it is more
It is secondary rinse well after, it is stand-by, 1.0-1.2ml gold chlorides are added in 120ml ultra-pure waters, are heated to fluidized state, Ran Hou
2.4-2.8ml three citric acid monohydrate sodium are rapidly added under quick stirring, 18-25mins is persistently stirred, obtains claret
Nano gold spherical solution；
（2）The BSPP modifications of nano gold spherical：100ml round-bottomed flask is taken, the nano gold spherical solution that will be cooled to room temperature adds it
In, then add 1000-1100 times of BSPP, under room temperature condition, stand overnight, that is, obtain the stable nano gold sphericals modified of BSPP
Solution；
（3）The antibody modification of nano gold spherical：The BSPP nano gold sphericals modified are utilized into supercentrifuge, 15-25mins is centrifuged, removes
Fall liquid, be melted into the SDS solution of mass fraction 0.8%, be divided into two parts again, with antibody and nano gold spherical according to 1000:1-
1200:1 ratio is added primary antibody and secondary antibody respectively, is then gradually added into sodium chloride solution to 0.2M is finally reached, then
3-5 hours are placed, centrifugation removes antibody unnecessary in solution, is redissolved in 0.4X TBE solution, that is, obtains nanogold
The antibody modification solution of ball.
In the present invention, it is preferred to step（1）Middle gold chloride mass fraction is 2%, preferably the quality of three citric acid monohydrate sodium point
Number is 2%.
In the present invention, it is preferred to step（3）In, the centrifugal rotational speed of centrifuge is 8500-9000rcf.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of the technical principle of the present invention is not departed from, some improvement and modification can also be made, these improve and become
Type also should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of collaurum early pregnancy detection reagent, it is characterised in that the collaurum early pregnancy detection reagent is mainly by two parts group
Into being the golden standard liquid for linking primary antibody and secondary antibody respectively.
2. a kind of collaurum early pregnancy detection reagent as claimed in claim 1, it is characterised in that the link primary antibody and secondary antibody
Golden standard liquid is respectively golden standard liquid one and golden standard liquid two.
3. a kind of preparation method of collaurum early pregnancy detection reagent as claimed in claim 1, it is characterised in that including following step
Suddenly：
The preparation of nano gold spherical：The used glass apparatus of experiment uses chloroazotic acid（Hydrochloric acid：Nitric acid=3:1）Immersion 4 hours, repeatedly
It is stand-by after rinsing well, 0.8-1.0ml gold chlorides are added in 100ml ultra-pure waters, fluidized state are heated to, then fast
2.0-2.4ml three citric acid monohydrate sodium are rapidly added under fast stirring, 15-20mins is persistently stirred, obtains claret and receive
Meter Jin Qiu solution；
The BSPP modifications of nano gold spherical：100ml round-bottomed flask is taken, the nano gold spherical solution that will be cooled to room temperature is added thereto,
Then add 500-1000 times of BSPP, under room temperature condition, stand overnight, that is, it is molten to obtain the stable nano gold sphericals modified of BSPP
Liquid；
The antibody modification of nano gold spherical：The BSPP nano gold sphericals modified are utilized into supercentrifuge, 10-20mins is centrifuged, removes
Liquid, it is melted into the SDS solution of mass fraction 0.5%, is divided into two parts again, with antibody and nano gold spherical according to 100:1-1000:1
Ratio be added primary antibody and secondary antibody respectively, be then gradually added into sodium chloride solution to 0.1M is finally reached, then place 3-5
Hours, centrifugation remove antibody unnecessary in solution, are redissolved in 0.2X TBE solution, that is, obtain the antibody of nano gold spherical
Modify solution.
4. the preparation method of collaurum early pregnancy detection reagent as claimed in claim 3, it is characterised in that the step（1）In
Gold chloride mass fraction is 1%, and the mass fraction of the three citric acid monohydrates sodium is 1%.
5. the preparation method of collaurum early pregnancy detection reagent as claimed in claim 3, it is characterised in that the step（3）In,
The centrifugal rotational speed of centrifuge is 5000-8000rcf.
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| CN201710833041.2A CN107843736A (en) | 2017-09-15 | 2017-09-15 | A kind of collaurum early pregnancy detection reagent and preparation method thereof |
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| CN201710833041.2A CN107843736A (en) | 2017-09-15 | 2017-09-15 | A kind of collaurum early pregnancy detection reagent and preparation method thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090181097A1 (en) * | 2005-11-29 | 2009-07-16 | Stevens Molly M | Particles |
| CN101821617A (en) * | 2007-09-17 | 2010-09-01 | 浦项工科大学校产学协力团 | Solid substrate and production and preparation method thereof with molecule of surface combination |
| CN201897595U (en) * | 2010-11-16 | 2011-07-13 | 上海英伯肯医学生物技术有限公司 | Rapid and visual early pregnancy reagent box |
| CN104962615A (en) * | 2015-06-04 | 2015-10-07 | 西南大学 | DNA flexagon nano structure-nanogold biosensor based on adapter modification and preparing method and application of DNA flexagon nano structure-nanogold biosensor |
| CN107058505A (en) * | 2017-02-17 | 2017-08-18 | 四川农业大学 | One kind visualization staphylococcus aureus detection nano-probe and preparation method thereof |
-
2017
- 2017-09-15 CN CN201710833041.2A patent/CN107843736A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090181097A1 (en) * | 2005-11-29 | 2009-07-16 | Stevens Molly M | Particles |
| CN101821617A (en) * | 2007-09-17 | 2010-09-01 | 浦项工科大学校产学协力团 | Solid substrate and production and preparation method thereof with molecule of surface combination |
| CN201897595U (en) * | 2010-11-16 | 2011-07-13 | 上海英伯肯医学生物技术有限公司 | Rapid and visual early pregnancy reagent box |
| CN104962615A (en) * | 2015-06-04 | 2015-10-07 | 西南大学 | DNA flexagon nano structure-nanogold biosensor based on adapter modification and preparing method and application of DNA flexagon nano structure-nanogold biosensor |
| CN107058505A (en) * | 2017-02-17 | 2017-08-18 | 四川农业大学 | One kind visualization staphylococcus aureus detection nano-probe and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| TAO HU 等: "Colorimetric sandwich immunosensor for Aβ(1-42) based on dual antibody-modified gold nanoparticles", 《SENSORS AND ACTUATORS B》 * |
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Application publication date: 20180327 |